JFS Abstract Details

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PUFAs in Fish: Extraction, Fractionation, Importance in Health

Abstract

Original Articles PUFAs in Fish: Extraction Fractionation Importance in Health F Sahena 1 ISM Zaidul 1 S Jinap 1 N Saari 1 HA Jahurul 1 KA Abbas 1 and NA Norulaini 1 1 Authors Sahena Zaidul Jinap Saari Jahurul and Abbas are with Faculty of Food Science and Technology Univ Putra Malaysia 43400 UPM Serdang Selangor DE Malaysia Author Norulaini is with School of Distant Education Univ Sains Malaysia 11800 USM Pulau Pinang Malaysia Direct inquiries to author Zaidul (E-mail: zaidul@foodupmedumy) Introduction Fish oils are a readily available source of long-chain polyunsaturated fatty acids (PUFAs) especially those of the n-3 series mainly cis-58111417-eicosapentaenoic acid (EPA; C20:5) and cis-4710131619-docosahexaenoic acid (DHA; C20:6) (Table 1) Recognition of the roles played by these fatty acids in human health and nutrition (Bang and Dyerberg 1972; Braden and Carrol 1986; Simopoulos 1991) and the resulting growth in new markets have stimulated much interest in methods of extracting and concentrating them from natural sources These PUFAs occur as triglycerides (TG) also called triacylglycerols in fish oils at levels between 10 and 25 (Haraldsson and others 1995) The nature and quantity of fish lipids vary according to species and habitats (Shamsudin and Salimon 2006) It is well known that fish lipids are the main sources of PUFAs especially EPA and DHA (Osman and others 2001) These 2 fatty acids cannot be synthesized by the human body and must be obtained from the diet (Linko and Hayakawa 1996) Fish oil is the main source of -3 fatty acids therefore the fatty acid composition of fish oil is very important for understanding its functional properties (von Schacky and others 1999; Nestel 2000) and in particular the correct ratio between -3 and -6 fatty acids which is very important Fish oils typically contain unsaturated straight-chain fatty acids ranging from C14 to C22 having from 1 to 6 double bonds Fish oil derivatives in the form of -3 fatty acids are increasingly demanded as pharmaceutical products food additives and dietary health supplements Interest in -3 fatty acids (such as C18:3 alpha-linolenic acid ALA) C20:5 eicosapentaenoic acid (EPA) and C22:6 docosahexaenoic acid (DHA) began several years ago and now there is an extensive scientific literature supporting their positive role in human health because they can intervene in the prevention and modulation of certain diseases that are common in many populations; research studies have shown that -3 can reduce the risk of heart disease and high blood pressure prevent blood clots protect against cancer and even alleviate depression (Shahar and others 1994; von Schacky and others 1999) Among the compounds of interest are concentrates of EPA (5cis 8cis 11cis 14cis 17cis eicosapentaenoic acid) and DHA (4cis 7cis 10cis 13cis 16cis 19cis docosahexaenoic acid) (Table 1) They have pharmaceutical value in the prevention of atherosclerosis heart attack hypertension and cancer (FAO 1998) Clinical trials have shown that fish oil supplementation is effective in the treatment of many disorders including rheumatoid arthritis (Kremer 2000) and diabetes Also results of clinical and epidemiological research suggest that EPA and DHA found only in fish and seafood have extremely beneficial properties for the prevention of human coronary artery disease (Leaf and Weber 1998) Along with this knowledge an increasing interest in suitable commercial processes to recover PUFAs in concentrated form can be monitored The conventional methods for their extraction fractionation and purification include vacuum distillation urea crystallization hexane extraction and conventional crystallization all having the disadvantages of requiring high-temperature processing resulting in loss or decomposition of the thermally labile compounds or employing flammable or toxic solvents (Staby and Mollerup 1993) Therefore extraction with supercritical fluids as solvents especially carbon dioxide offers new opportunities for the solution of separation problems (Brunner and Riha 2000) Supercritical carbon dioxide is a promising solvent for the extraction and fractionation of edible oils containing labile PUFAs Extraction can be carried out at lower pressure and lower temperature Besides supercritical fluid extraction (SFE) offers new opportunities thanks to the fact that CO2 is a nontoxic nonflammable inexpensive and clean solvent However the purposes of this review were to identify the higher content of PUFAs especially EPA and DHA in cheaper fish sources and their better methods of separation purification and fractionation Chemistry Polyunsaturated fatty acids (PUFAs) are classified according to the position of double bonds either from the methyl end or the carboxyl end In the omega or n convention the double bonds are counted from the methyl end of the carbon chain A majority of PUFAs of biological importance belong to the -6 (arachidonic acid) and -3 (eicosapentaenoic acid) groups The 1st double bond in -3 fatty acids starts at the 3rd carbon atom from the methyl end The fatty acids belonging to this family are -linolenic acid (ALA 18:3) eicosapentaenoic acid (EPA 20:5) docosahexaenoic acid (DHA 22:6 -3) and docosapentaenoic acid (DPA 22: 5) The chemical structures of these fatty acids are shown in Figure 1 Due to nonconjugated (methylene interrupted) cis-type double-bond configuration the carbon chain is bent at the site of double bonds and the molecule shows some crumpling These fatty acids originate in unicellular phytoplanktons and seaweeds and accumulate in fish (Ackman 1980) The precursor of long-chain -3 fatty acids is ALA which is subsequently chain-elongated and desaturated to EPA and DHA Both EPA and DHA occur as constituents of fish triglycerides and phospholipids Figure 2 shows the pathway of omega-3 and omega-6 fatty acid synthesis where the PUFAs are either n-3 or n-6 fatty acids ALA and linoleic acid are the precursors of n-3 and n-6 fatty acids respectively and are converted to different long-chain PUFAs by sequential desaturation and elongation PUFAs in Disease The importance of a balanced PUFA intake has been recognized by health organizations throughout the world over the past decade There is now some consensus that PUFAs should form a bare minimum 3 and preferably 10 to 20 of the total lipid intake and that the 6- to 3-series ratio should ideally be around 4 : 1 or 5 : 1 Although the biological effects of eicosanoids are undisputed the same can not be said for their PUFA precursors and most of the diverse pharmacological effects that have been proposed for PUFAs have yet to be proved Also the importance of PUFA intakes as compared with other nutritional factors genetic determinants and environmental influences in the initiation and progress of diseases is not clear The pioneering epidemiological work of Dyerberg and Bang (1979) and Dyerberg (1986) on Greenland Eskimos suggested a possible link in low incidence of heart diseases to the consumption of seafood Since then many studies have been published on the role of -3 fatty acids in human health and diseases Pamela (2001) reported that EPA and DHA have biochemical effects in the prevention and treatment of several disorders and diseases such as coronary heart disease rheumatoid arthritis asthma cancers diabetes and others At present the major sources of EPA and DHA are from the cool deep-sea fish oil such as menhaden cod sardine anchovy and others (Shamsudin and Salimon 2006) These fatty acids have been reported to have beneficial effects in cardiovascular diseases autoimmune disorders and various inflammations (Weaver and Holub 1988) The beneficial effects of -3 fatty acids are explained through altered zicosanoid metabolism in the circulatory system leading to production of prostaglandins considerably weaker in inducing platelet aggregation than those produced from -6 fatty acids The physiological effects of -3 fatty acids are: (1) lowering of plasma triglycerides; (2) increased aggregation time for platelets; (3) decreased viscosity of blood; (4) decreased blood pressure; (5) reduction of arthrosclerosis; (6) reduction of inflammation arthritis psoriasis asthma; (7) reduction in tumors Prevention of cancer is still debated as unsaturated fatty acids produce free carbonyl compounds which are tumorigenic Detailed treatment of the medical aspects has been the subject of many publications (Kinsella 1986 1987 1990; Lees and Karel 1990) An increase in PUFA consumption carries an elevated risk of exposure to toxic oxidation products which are implicated in cancer and thrombotic and inflammatory diseases In particular there is concern that current intakes of dietary antioxidants are insufficient to cope with an increased polyunsaturate load and that serious health hazards could be posed by an elevation of in vivo PUFA-peroxide production Hence any moves designed to increase dietary polyunsaturates must be accompanied by measures to ensure that antioxidant levels are adequately maintained PUFAs in Human Health PUFAs are long-chain fatty acids containing 2 or more double bonds The potential applications of PUFAs are found in therapeutics foods and nutrition They can be found in animals plants algae fungi and bacteria They are produced commercially from selected seed plants and some marine sources PUFAs are grouped into 2 series on the basis of the position of the terminal double bond being 3C or 6C from the terminal carbon atom of the fatty acid chain Some examples are: 3-series PUFA linolenic acid (LA) and alpha-linolenic acid (ALA) Six-series PUFA gamma-linolenic acid (GLA) arachidonic acid (AA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) They provide structural and functional characteristics and are involved in a wide range of biological components including membranes phospholipids They are involved in regulating architecture dynamics phase transitions and permeability of membranes and control of membrane-associated processes Also they are involved in regulating membrane-bound proteins such as ATPase transport proteins and histocompatibility complexes In addition PUFAs regulate expression of some genes including those coding for fatty-acid synthase nitric-oxide synthase and sodium-channel proteins Thus they have an impact on cellular biochemical activities transport processes and cell-stimulus responses They are involved in physiological processes including immune responses and cold adaptation and are implicated in pathological conditions such as cardiovascular disease Equally important PUFAs serve as precursors for conversion into metabolites that regulate critical biological functions In plants they are transformed by a variety of enzymes into oxygenated molecules ranging from simple aldehydes to oxylipins These act as anti-infection agents wound-response mediators and regulatory hormonal and chemotactic agents as well as aroma and taste compounds In humans PUFA metabolism and eicosanoid function became important when it was discovered that arachidonate is the precursor for prostaglandins Ecosanoids are a diverse group of hormones including prostaglandins thromboxanes and leukotrienes Research shows that eicosanoid hormones are fundamental to proper maintenance of homeostasis and are linked to important physiological and pathophysiological conditions Figure 3 shows that the DHA from the blood or the conversion of linolenic acid (LNA) in the liver may be esterified in phosphatidylcholine (PC) and secreted in lipoproteins as PC-DHA which can be converted into lysoPC-DHA by the endothelial lipase (Lagarde 2008) It can be speculated that lysoPC-DHA might alternatively be produced in and secreted by the liver LysoPC-DHA is taken up by the brain to release DHA or for being converted into PC-DHA with DHA redistributed within other phospholipids (Lagarde 2008) Author also reported that DHA the end-product of the n-3 family fatty acid is an abundant component in the brain phospholipids and a major nutrient of marine lipids DHA is also a fairly good substrate of lipoxygenases especially the n-9 and n-6 ones Hydroxy derivatives that is docosanoids exhibit potent biological activities which may explain part of the potential benefit of DHA in the brain and vascular bed (Lagarde 2008) The eicosanoid pathway in mammals begins with the phospholipase-mediated release of PUFAs from membrane phospholipids and is followed by cyclooxygenase-catalyzed reactions that give rise to such major classes of metabolites as prostaglandins thromboxanes lipoxins and leukotrienes In 1971 John Vane discovered that asiprin inhibits the cyclooxygenase enzyme and thus inhibits prostaglandin synthesis This is the basis for aspirin as an anti-inflammatory agent Also prostaglandins and thromboxanes are involved in regulating blood clot formation so baby aspirins are prescribed for patients at risk of heart diseasestroke because a low dose of aspirin disturbs the PGTX balance and inhibits clot formation Present-day eicosanoid-related ailments can be traced to changes in human nutrition Research indicates that intake of PUFAs was evenly balanced 10000 years ago with 3- and 6-series PUFAs Since then reductions in 3-series PUFAs have created an imbalance in most modern societies This can be attributed to a move away from huntergatherer nutrition resulting in a large rise in 6-series PUFA intake from the increased consumption of particular plants and animal products from intensive agriculture which has reduced levels of other 3-series PUFA types Church and others (2008) summarized that excess and deficient dietary -3 fatty acids during pregnancy and lactation caused delayed neural transmission along the brainstem auditory pathway as evidenced by prolonged auditory brainstem response (ABR) latencies in a 24-d-old rat offspring The excess offspring also showed postnatal growth retardation and a trend for increased postnatal mortality Their study's investigation of both deficient and excess -3 fatty acids consumption during pregnancy and lactation has important health implications Current United States consumption of -3 fatty acids is lower than national and international recommendations (Akabas and Deckelbaum 2006) and excess -3 fatty acids is being consumed voluntarily (Rump and others 2001; Thorsdottir and others 2004) and being given as treatment for preterm birth (Sattar and others 1998; Smuts and others 2003) Church and others (2007 2008) also reported that both excess and deficient amounts of dietary -3 fatty acids during pregnancy and lactation can cause postnatal growth retardation as well as sensory and neurological abnormalities in the offspring Even though supplementing one's diet with modest amounts of -3 fatty acids can be beneficial consuming or administering large amounts of -3 fatty acids to pregnant women or nursing infants could have harmful consequences Despite such caveats some recent publications are still recommending that pregnant women and infants should consume higher than normal amounts of -3 fatty acids (Akabas and others 2006; Jensen 2006) Church and others (2008) disagree with this above statement Moreover recent literature reviews have failed to find convincing evidence that prenatal fish oil treatments improve pregnancy outcome (Makrides and others 2006) and failed to find that postnatal -3 fatty acids supplementation during nursingbottle-feeding has lasting effects on infant brain and visual functions (Hadders-Algra and others 2007) In light of such findings and because excess -3 fatty acids during pregnancy and lactation can have adverse neurological and developmental effects we conclude that using large amounts of dietary -3 fatty acids during pregnancy and lactation has little merit is potentially harmful to the offspring and should not be advocated in clinical or public practice Instead more research is needed to determine: (1) how much perinatal -3 fatty acids is too much too little and just right for the developing human and (2) the long-term consequences with regard to the fetal programming of neurodevelopmental and sensory impairments and the adult-onset diseases of hypertension diabetes age-related neural degeneration and a shortened life span (Barker 2004) n-3 PUFAs exert many of their beneficial effects upon the cardiovascular system via their effects on several cellular processes n-3 PUFAs improve the plasma lipid profile Siddiqui and others (2008) reported that intake of n-3 PUFAs (4 gd for 6 wk) reduced TG levels by 18 to 20 but had a minimal impact on low-density lipoprotein cholesterol or high-density lipoprotein cholesterol (HDL-C) (Mori and others 2000) In contrast to these studies long-term treatment of hypertriglyceridemic patients with n-3 PUFAs (4 gd for 16 wk) led to a significant reduction in TG by 47 while TG levels rose by 16 with placebo (corn oil) This effect of n-3 PUFAs was associated with a decrease in total cholesterol: HDL ratios (20) and a modest increase in HDL-C (13) (Harris and others 1997) Similar results were also reported in another study where hypertriglyceridemic patients were treated with n-3 PUFAs (4 gd) for 6 mo (Abe and others 1998) It appears from different studies (McKenney and Sica 2007) that higher levels of n-3 PUFAs for longer duration have beneficial effects on plasma lipid profile n-3 PUFAs also have antiatherogenic actions Eritsland and others (1996) reported that n-3 PUFA supplementation (4 gd for 1 y) in postcoronary artery bypass graft patients was associated with a reduced frequency of vein graft occlusions This n-3 PUFA effect was not linked to an influence on serum lipoproteins because serum cholesterol levels were not altered by n-3 PUFA supplementation Furthermore there was no association between the reduction in serum TG and vein graft occlusion These studies therefore concluded that the n-3 PUFA effect on new plaque development appeared to be due to antithrombotic as well as antiatherosclerotic properties of n-3 fatty acids Furthermore Thies and others (2003) reported that n-3 PUFA supplementation (1 to 4 gd for an average of 42 d) in heart patients prior to undergoing carotid endarterectomy resulted in a rapid incorporation of n-3 PUFAs into advanced atherosclerotic plaques which was associated with structural changes consistent with increased plaque stability The antiatherosclerotic effects of n-3 PUFAs appear to be mediated through their anti-inflammatory effects on platelets and endothelial cells Platelets through their interaction with the vascular endothelium play a critical role in atherogenesis (Ross 1993) Mori and others (1997) observed that human consumption of n-3-PUFAs (3 to 4 gd) for 3 wk reduced platelet aggregation induced by collagen and platelet-activating factor (PAF) regardless of whether n-3 PUFAs were ingested as daily fish meals or fish oil capsules Similarly Agren and others (1997) reported that consuming moderate amounts of n-3 PUFAs for 15 wk in the form of a fish diet (038 g EPA 067 g DHAd) or fish oil (133 g EPA 095 g DHA) also inhibited platelet aggregation but did not affect hemostatic factors Notably EPA-free DHA oil (168 gd) was not effective in decreasing in vitro platelet aggregability (Agren and others 1997) The ineffectiveness of DHA implies that modulation of platelet aggregation by n-3 PUFAs may be mediated through the eicosanoid pathway rather than being a direct effect of fatty acids on platelets Furthermore in the patients with elevated TG levels prolonged treatment with n-3 PUFAs (4 gd for 7 mo) was associated with reduced levels of soluble adhesion molecules (sICAM-1 and sE-selectin) (Abe and others 1998) Soluble adhesion molecules lack membrane-spanning and cytoplasmic domains that are present in the membrane-bound forms but their levels have been noted to be elevated in pathological conditions in which tissue expressions of the membrane bound forms of adhesion molecules are known to be unregulated (Ballantyne and others 1994) It is difficult to measure the membrane expression of adhesion molecules in the human vasculature after n-3 PUFA supplementation Evidence for n-3 PUFA effects on cell membrane expression of adhesion molecules is derived from in vitro experiments DHA treatment to human adult saphenous vein endothelial cells at concentrations (10 M) compatible with nutritional supplementation of this fatty acid to individuals consuming a normal Western diet reduced surface expression of adhesion molecules (De Caterina and others 2000) It appears from these studies that one of the beneficial effects of n-3 PUFAs on the cardiovascular system is mediated through its antiatherosclerosis properties Furthermore n-3 PUFAs also improve vascular functions However dietary supplementation with fish oil (5 g EPADHAd for 3 wk) significantly improved endothelium-dependent coronary vasodilation in heart transplant recipients without altering the responses to endothelium-independent vasodilation These improved vascular functions play a small role in reducing hypertension A metaregression analysis of 36 randomized trials on fish oil supplementation (mean consumption of 37 gd for a median of 12 wk) in largely overweight and hypertensive subjects showed only a small antihypertensive effect (Geleijnse and others 2002) Furthermore it is suggested that DHA is likely more favorable in lowering blood pressure and heart rate than EPA (Mori 2006) In conclusion observational studies human intervention trials animal models and cell culture studies suggest that n-3 PUFAs have beneficial effects on the cardiovascular system The molecular and cellular effects of n-3 PUFAs for mediating the beneficial cardiovascular effects are not fully known Siddiqui and others (2008) reported that the beneficial effects of fish oil are attributed to their n-3 polyunsaturated fatty acid (PUFAomega-3 fatty acids) content particularly EPA (20:5 n-3) and DHA (22:6 n-3) Dietary supplementation of DHA and EPA influences the fatty acid composition of plasma phospholipids that in turn may affect cardiac cell functions in vivo Recent studies have demonstrated that long-chain omega-3 fatty acids may exert beneficial effects by affecting a wide variety of cellular signaling mechanisms Pathways involved in calcium homeostasis in the heart may be of particular importance L-type calcium channels the NaCa2 exchanger and mobilization of calcium from intracellular stores are the most obvious key signaling pathways affecting the cardiovascular system; however recent studies now suggest that other signaling pathways involving activation of phospholipases synthesis of eicosanoids regulation of receptor-associated enzymes and protein kinases also play very important roles in mediating n-3 PUFA effects on cardiovascular health There is direct evidence that suggests n-3 PUFAs are very potent agents in regulating intracellular calcium levels Earlier studies have shown that EPA and DHA (5 M) can prevent arrhythmias fibrillation and contracture in isolated rat cardiac myocytes induced by toxic concentrations of ouabain (Hallaq and others 1990) a cardiac glycoside that binds to the -subunit of membrane-bound Na K-ATPase (Wallick and others 1974) Addition of either oxygenase inhibitors or antioxidants did not alter the effects of n-3 PUFAs on ouabain-induced cardiac arrhythmia (Hallaq and others 1990) This observation suggests that n-3 PUFA incorporation into the phospholipids of cell membranes may have prevented the toxicity caused by ouabain and its presence was associated with fewer rises in cytosolic free calcium (Hallaq and others 1990) Furthermore EPA (2-10 M) exhibited a similar protective effect against ouabain toxicity when cardiomyocytes were incubated for 3 to 5 d in the presence of these n-3 PUFAs (Hallaq and others 1992) Two other PUFAs linoleic acid (18:2 n-6) and linolenic acid (18:3 n-3) also exhibited similar but less potent effects compared with EPA In contrast neither oleic acid (18:1 n-9) nor saturated fatty acids (18:0 14:0 12:0) affected contraction rate (Hallaq and others 1992) These studies have shown that the beneficial effects of fish oil in preventing fatal arrhythmias in myocardial ischemia are at least in part mediated by modulating the dihydropyridine-sensitive L-type calcium current (Hallaq and others 1992) Consistent with this observation treatment of cardiomyocytes with DHA or EPA prevented the increase in calcium influx by Bay K8644 an established agonist for L-type calcium channels (Hallaq and others 1992) strongly suggesting that L-type calcium channels are the target site for these fatty acids Similarly it has been demonstrated that n-3 PUFAs modulate calcium current through the L-type calcium channels and these effects occur within minutes of adding EPA or DHA to the perfusing medium of the cultured cardiac myocytes (Leaf 1995) Moreover it has also been shown that the delayed rectifier K channel is inhibited by n-3 PUFAs (Honore and others 1994) The combined effect of this is suggested to reduce electrical excitability making arrhythmias less likely (Kang and others 1995) Both EPA and DHA are known to be antiarrhythmic They depress surface membrane electrical excitability (Kang and others 1995) and inhibit spontaneous release of Ca2 from overloaded cardiac SR (Negretti and others 2000) The effect of n-3 PUFAs on the L-type Ca2 channel appears to be due to their direct binding to the channel proteins This fact is supported by a recent study investigating the link between n-3 PUFA content of the plasma membrane and ion channel activity (Pound and others 2001) which suggested that n-3 PUFA concentrations required for antiarrhythmic action were too low to produce a significant change in the overall arrangement of the phospholipids within cardiac membranes Similarly the effect is quickly reversed when free PUFAs are extracted from the cells by adding dilapidated BSA to the bathing medium (Kang and Leaf 1996) These observations imply that n-3 PUFAs are neither fully incorporated into membrane phospholipids nor covalently bound to any constituents of the myocyte to produce the antiarrhythmic effect (Kang and Leaf 1996) These studies therefore suggest that n-3 PUFAs exert antiarrhythmic effects by direct interactions with SL ion channels rather than indirectly by perturbing membrane phospholipids packing Pharmaceutical and Food Applications Evidence of the possible medical effects of PUFA deficiencies have coupled with the growing acceptance of pharmafoods by consumers brought these compounds to the attention of food and pharmaceutical companies which have been quick to exploit markets in the biomedical and pharmafood areas A variety of specialty PUFA lipids are available for uses ranging from antiaging antithrombotic anti-inflammatory anticholesterolemic and anticancer drugs to immunostimulant and immunosuppressant therapeutics However their efficiencies remain to be verified Unregulated applications for esters glycerides and phospholipids such as health food additives for foods nutritional formulas and cosmetics ingredients have also increased The most obvious commercial impact of PUFAs has been in health supplements with a host of plant- and fish-derived GLA EPA and DHA products now being available in the marketplace for uncontrolled dietary use This raises a question whether PUFAs should be treated as exceptional nutritional products or as pharmaceuticals As they occur in foodstuffs there is a case for considering them as generally recognized as safe food components; on the other hand they are biologically active In view of the serious disease states that may result from eicosanoid imbalances there is justification to regard them as biomedical additives Despite limited understanding of PUFA biochemistry and many therapeutic nutritional and regulatory issues that remain to be resolved the market potential for PUFA in the food health-supplement cosmetics and pharmaceutical sectors is obvious In addition to such first generation applications it is predicted that high-value PUFA-derived eicosanoids aimed at treating specific disease conditions will appear in the therapeutics market once biotechnological approaches are in place for their production PUFAs in Fishes Mishra and others (1993) reported that the total world production of fish oil in 1988 was 15 million metric tons (FAO Yearbook of Fishery Statistics; Commodities 1988) Most of this production has been used in various food and pharmaceutical formulations Typical food uses of hydrogenated or partially hydrogenated fish oils are production of salad oils frying oils table margarines low-calorie spreads and shortenings in bakery products (Bimbo 1990; Bimbo and Crowther 1991) Most of the production from the United States is exported overseas mainly to Europe which is the major fish oil consumer Currently it is known that fatty acids of fish flesh are the most beneficial for human health due to its high proportion of unsaturated fatty acids Fish lipids are well known to be rich in long-chain n-3 PUFAs especially EPA and DHA (Osman and others 2007) Research has also indicated that fatty acid composition of the fish differs due to climatic influence diet age maturity and type of species (Kinsella 1988; Ratkowsky and others 1996; Saito and others 1999; Halilolu and others 2004; elik and others 2005) Fish from tropical climate were found to have lower amounts of total lipids compared to fish from the arctic region Besides the lipids of freshwater feeds are characterized by linoleic (C18:2 n-6) and linolenic (C18:3 n-3) acids and EPA (Halilolu and others 2004; elik and others 2005) The plankton of marine feeds presents low levels of n-6 PUFA of which EPA and DHA are the predominant acids (Justi and others 2003) Thus marine fish are distinguished by high concentrations of n-3 since they feed on plankton while freshwater fish mainly contains n-6 fatty acids The fatty acid compositions of some marine fishes are listed in Table 2 (Osman and others 2007) The most abundant PUFA in the fin fishes (head middle and tail) was C22:6 (DHA) (818 to 2395) while C20:5 (EPA) was also present in significant proportion (713 to 1961) with n-6 fatty acids also present in significant proportion C18:2 n-6 ranged from 547 to 1611 while C20:4 n-6 had a range from 034 to 1341 (Table 2) However Suriah and others (1995) reported that the make-up of fatty acids of fin fish from seawater is slightly different when compared to that of freshwater fish where the concentrations of MUFAs are higher than the saturated and PUFAs Other researchers have also reported that freshwater fish have lower contents of PUFAs (Vlieg and Body 1988) The differences can be due to the fact that freshwater fishes feed mainly on vegetation and plant materials while marine fishes feed on zooplanktons which are rich in PUFAs According to Piggott and Tucker (1990) the n-6 : n-6 ratio is a better index in identifying nutritional value of fish oils of different species Osman and others (2007) reported that in terms of individual PUFA contents most of the fin fish analyzed had higher contents of AA and DHA than menhaden oil (Table 2) As for DHA content all of the fishes studied had higher percentages of DHA as compared to menhaden oil (79) The highest level was found in bagi (Aacnthurs nigrosis) (2395) which was 3 times higher than in menhaden oil However for the EPA most of the species studied had lower concentrations (751 to 1153) than the EPA concentration of menhaden oil (125) This finding is in agreement with the data reported by Osman and others (2001) and Wang and others (1990) All these researchers revealed that marine fish were rich in n-3 especially EPA and DHA Osman and others (2001) studied fatty acid composition of selected marine fish in Malaysian waters and have summarized the total fatty acid contents (Table 3) Authors reported that the PUFA contents were much higher (56 to 92) than the saturated fatty acids (363 to 114) and MUFAs were lowest (1 to 10) in marine fish The trend is different when compared to an earlier study on freshwater fish where the concentrations of MUFAs were higher than the saturated and PUFAs (Suriah and others 1995) Other researchers have also shown that freshwater fish have lower contents of PUFAs (Vlieg and Body 1988) The difference can be attributed to the fact that freshwater fishes feed largely on vegetation and plant materials whereas marine fish staple diets are mainly zooplanktons rich in PUFAs Menhaden oil which is marine fish oil has been proposed by the US FDA (1997) as a PUFA supplement The concentrations of -3 PUFA (297 to 484) and other PUFAs (277 to 40) in the study of FDA (1997) were found to be much greater than found in the standard menhaden oil (-3 PUFA 289; other PUFAs 166 whereas -6 PUFAs are 143) The values for MUFAs (137 to 912) and saturated fatty acids (363 to 1638) were lower than those of menhaden oil (MUFA 194; saturated 202) (Table 3) This study has shown that marine fish were richer in -3 PUFAs (297 to 484) than -6 PUFAs (11 to 20) Suriah and others (1995) O'Dea and Sinclair (1982) and Suzuki and others (1986) reported on freshwater and cultured fish had shown opposite results where the levels of -3 PUFAs were lower than -6 PUFAs However Wang and others (1990) reported similar findings in that marine fish were rich in -3 especially DHA and EPA The contents of arachidonic acid (AA) EPA and DHA of the fish analyzed ranged from 019 to 068 082 to 676 and 936 to 286 respectively Fish that had higher contents of AA than the menhaden oil (047) were striped sea catfish (068) silver pomfret (060) and black pomfret (052) Based on an earlier study (Suriah and others 1995) in some cases the contents of AA in marine fishes were lower than that of freshwater fishes The DHA contents of all the fish studied were higher than the amounts found in menhaden oil (620) and the highest level found in hardtail scad (286) was almost 5 times higher The amounts of EPA in the species studied (082 to 676) were lower than in the menhaden oil (854) However the other richest and cheapest source of PUFAs is Indian mackerel Charles and others (2003) chose mackerel waste to extract PUFAs because it is an underutilized fish with a limited range of value-added products developed from it This is probably because of the highly perishable nature of the species Being so oily makes the fish very prone to postmortem spoilage such as rancidity and presents processing challenges (Banks 1967) However in light of the nutritional value of marine oils the oily nature of mackerel tissues presents opportunities It is important to increase the information base available on mackerel to enhance its exploitation The baseline characteristics of mackerel oil in Table 4 indicate that the distribution of oil in the tissues is not proportional and the skin yields are the highest at about 38 compared to 9 for viscera and muscle This disparity was evident irrespective of the solvent extraction system used Consequently the use of chloroformmethanol (2 : 1 volvol) for oil extraction was discontinued after the preliminary analysis since there were no inherent advantages over hexaneisopropanol (3 : 2 volvol) that would justify the risk of toxicity associated with exposure to both chloroform and methanol Since mackerel skins are usually discarded during processing and they have high oil content skins are a most suitable material for producing PUFAs because they will generate comparatively high yields (about 38 ww) of oil for small inputs thus reducing bulk handling and transportation Indian mackerel are widespread in the Indo-West Pacific from South Africa Seychelles and Red Sea areas and east through Indonesia and off northern Australia to Melanesia Micronesia Samoa China and the Ryukyu Islands They have entered the eastern Mediterranean Sea through the Suez Canal Indian mackerel is a very important species Catches are usually recorded as Rastrelliger spp or combined with R brachysoma In the last 25 y the world catch for R kanagurta alone fluctuated between about 96000 t in 1975 and a peak of 351193 t in 1994; since 1984 catches reported to FAO as Rastrelliger spp have exceeded 300000 t annually In the Western Indian Ocean area most of the catches (about 185000 t in 1995) are identified as R kanagurta while in the Eastern Indian Ocean 224000 t are reported as Rastrelliger spp and 43000 t as R kanagurta Instead in the Western Central Pacific which ranks as the area of major catches for Rastrelliger species 252000 t are not identified at the species level 104000 t are identified as R kanagurta and 26000 t as R brachysoma Indian mackerel are caught with purse seines encircling gillnets high-opening bottom trawl lift nets and bamboo stake traps The total catch reported for this species to FAO for 1999 was 302387 t The countries with the largest catches were India (146367 t) and the Philippines (53606 t) Conventional Extraction of PUFAs from Fish The production of fish oil deals with the separation of fatty substances (lipids) from other constituents of the fish Generally separation starts from the preparation of the raw material up to the purification of the product which is the final stage of the process One of the methods used industrially in obtaining fish oil is hydraulic pressing; a batch process whereby the oil is obtained or expressed by hydraulic pressing a mass of moderately cooked oil-bearing fish A recent development is in the extraction of oil from oil-bearing material using a solvent or solvent system Solvent extraction which is also referred to as leaching is a process whereby soluble constituents present either as solids or liquids are removed by the use of solvents In fact solvent extraction techniques are among the most commonly used methods of isolating lipids from food samples (including fish) and of determining the total lipid content of foods The principle is based on the fact that lipids are soluble in organic solvents but insoluble in water hence providing a convenient method of separating the lipid components in the food samples from water-soluble components such as protein carbohydrates minerals and water itself (McClements 2003) For a successful extraction of oil a sample must undergo specific preparations prior to solvent extraction (McClements 2003) In practice the efficiency of solvent extraction depends on the polarity of the lipids present compared to the polarity of the solvent Polar lipids (such as glycolipids or phospholipids) are more soluble in polar solvents (such as alcohols) than in nonpolar solvents (such as hexane) On the other hand nonpolar lipids (such as triacylglycerols triglycerides) are more soluble in nonpolar lipid than in polar ones Soxhlet extraction is one of the most commonly used methods for determination of total lipids in dried samples It is fairly simple to carry out and is the officially recognized method for a wide range of fat content determinations The main disadvantages of the technique are that: a relatively dry sample is needed (to allow the solvent to penetrate) it is destructive and it is time-consuming (McClements 2003) Shamsudin and Salimon (2006) extracted fish lipids from the local marine fish aji-aji of Malaysian waters using a mixture of chloroform : methanol (2 : 1) by Soxhlet extraction according to Andrade and others (1995) and Saify and Akhtar (2003) The crude extract was purified by adding an aqueous 088 KCl solution in 8 : 4 : 3 proportions of chloroform : methanol : KCl following the extraction method of Nordback and others (1998) The authors found that aji-aji fish contain about 183 crude lipids and about 168 lipid matter after purification process The results showed that aji-aji fish is high in lipid content Among local marine fish species studied aji-aji fish were found to contain average values in lipid percentages ranging from 10 to 21 (Osman and others 2001) Shamsudin and Salimon (2006) also reported that PUFAs in the omega-3 class exhibit higher percentages than PUFAs in the omega-6 class in aji-aji fish oil DHA (about 8) contained in aji-aji fish oil is higher than EPA (only about 1) comparatively However their percentages are much lower compared to EPA (about 13) and DHA (about 126) contained in menhaden fish oil (cold water fish) Lipid contents and fatty acid compositions of marine fishes differ with regard to species sex age size reproductive status geographic location and season (Piggott and Tucker 1990) Fish oil is a major source of PUFAs mainly EPA and DHA; and they are important for human health and nutrition as well as for the fish itself The role of PUFAs in fish is to maintain cellular structure and function and also to regulate normal cell growth and development (Cejas and others 2004) Under normal circumstances fish obtains the PUFAs from the diet mainly from phytoplankton Several methods have been reported for concentrating PUFAs in marine oils with varied yields Among methods that concentrate PUFAs as TGs without prior hydrolysis are solvent fractionation winterization and molecular distillation (Haraldsson and others 1995) Concentrations of up to 30 EPA and DHA are feasible using these methods However higher levels (65 to 80) are attainable by processes combining either hydrolysis or esterification with methods such as supercritical fluid extraction urea complexation and molecular distillation Concentrations beyond 90 are possible with high-performance liquid chromatography (HPLC; Haraldsson and others 1995) Urea complexation has been applied extensively to concentrate PUFAs from various sources including marine and vegetable oils (Ackman and others 1988; Traitler and others 1988; Hayes and others 1998; Ju and others 1998) Ratnayake and others (1988) demonstrated pilot-scale (20 kg) urea complexation for concentrating n-3 PUFAs Compared with the other methods for producing PUFA concentrates urea fractionation allows handling of large quantities of materials in simple equipment Since the process requires only limited use of less toxic organic solvents such as ethanol it is environmentally friendly It is also cost-effective because urea is relatively inexpensive Urea forms complexes with molecules containing linear alkyl chains which act as a template with which urea molecules complex in spiral-shaped structures during several hours of cooling (Hayes and others 1998) Separation of urea complexes from the nonurea complexing fraction effectively removes saturated and long-chain MUFAs and enriches the liquid extract in unsaturated FAs Fish processing could generate wastes of up to about 50 of the body weight of the processed fish based on the body components of interest to the processor (Babbit 1990) Wastes from fish processing are used to produce fishmeal with oil as a by-product or to remediate soil The oil content of fish waste lies between 14 and 401 (Babbit 1990) depending on the species and tissue Fish processing waste is therefore an important source of fish oil that could serve as a good source of PUFAs while adding value to the waste This study was undertaken to extract and characterize the oil from mackerel processing waste which is comprised mainly skin viscera and muscle tissues and to assess the possibility of concentrating PUFAs from the oil extracted from these tissues using urea complexation However conventional fish processing for removal of oil from proteins involves cooking pressing andor liquid extraction Removal of lipids with organic solvents causes protein denaturation and loss of functional properties (Pariser and others 1978) Adeniyi and Bawa (2008) extracted mackerel (Scomber scrombrus) oil using standard medium fish that were pretreated by washing drying particle size reduction and acid hydrolysis before transferring into the Soxhlet extraction apparatus for continuous extraction with petroleum ether (Macrae and others 1993) Refining of the extracted crude oil was carried out through degumming neutralization deodorization filtering and bleaching using methods as outlined by Hall (1992) The refined oil was then characterized to determine the moisture content and values of acid saponification iodine peroxide refractive index and specific gravity by employing the methods specified by Intl Standards Organization (ISO 1975 1988 1998) Graham and others (2007) reported that current omega-3 long chain-PUFA sources (predominantly oceanic fish oils) are in serious decline; moreover environmental contamination of marine environments has resulted in the presence of potentially toxic substances such as heavy metals and dioxins in fish oils Thus there is a very obvious requirement for an alternative and sustainable source of fish oils for use in human nutrition Enzymatic Extraction Mahmoud and others (2008) fractionated lipids from rainbow trout (Oncorhynchus mykiss) roe were extracted by chemical and enzymatic methods Figure 4 shows a scheme for the procedure of enzymatic extraction of trout roe The authors reported enzymatic hydrolysis by Alcalase Neutrase and Protamex yielded 3 fractions after centrifugation: a light phase (oil) an intermediate fraction (water-soluble material) and a residual heavy phase (Figure 4) The hydrolysis was performed within 120 min with the 3 tested enzymes and the degree of hydrolysis was calculated according to the pH-Stat method Using the different proteases some discrepancies were noticeable among the hydrolysis degree (DH) values Alcalase was the most efficient (78 DH) compared to Neutrase (32) and Protamex (23) Nevertheless the total nitrogen contents of the water phase were in the same range for Alcalase and Protamex indicating that Alcalase may lead to a high quantity of small peptides while Protamex and Neutrase may cut larger peptide fragments Moreover the DH from Alcalase on fish roe (78) can be compared with the 19 obtained with the same enzyme in trout fillets The low yield of fish roe proteolysis could lie in the specific composition of the chorion which must resist fungal and microbial attacks for days in water Also the insolubility of the chorion might be due to the formation of isopeptide bonds involving the side chains of Glu and Lys or Arg residues of its constituent proteins Heavy fractions of molecular mass of more than 2000 gmol represented about 25 of total peptides of the hydrolysate of trout roe treated with Alcalase Another remarkable result concerned the oil release after the different enzymatic extractions Alcalase remained the best tool for oil release with 385 of total lipid content compared to 296 and 183 for Protamex and Neutrase respectively Such observation is consistent with the fact that a high degree of hydrolysis is needed for a good oil extraction The authors also compared the melting points and enthalpies of the oils fractions obtained by solvent extraction against enzymatic processing including the supernatant neutral oil and the lipids contained in the heavy phase Melting points stood around 15 to 18 C indicating that the lipid fraction contained a high proportion of unsaturated fatty acids assessed by the low enthalpies of fusion which are characteristic of low-melting oils However it is interesting to notice that the melting point of the total oil extracted by solvents was an average of that of the lipids contained in the supernatant oil and in the heavy phase after enzymatic treatment This indicated that there is a partition of the oil composition between both phases during the enzymatic treatment Considering the enzymatic extraction it can be stressed that the supernatant oil is mainly composed of neutral lipids (975) while the lipids contained in the heavy phase are mainly polar lipids (733) This is probably due to their amphipatic properties that facilitate their linkage with hydrophilic material The analysis of the polar lipid fraction resulting from thin layer chromatography (TLC)-flame-ionization detector (FID) determinations also showed that solvents do not extract the same amounts of major phospholipids than a protease While both solvent methods released similar amounts of phosphatidylcholine (PC) phosphatidylethanolamine (PE) and phosphatidylinositol (PI) it appears that the major phospholipid released by Alcalase was mainly PC accounting for 896 of total polar lipids As the enzymatic process was carried out at pH 8 with no pH lowering at the end of the reaction time a positive charge could persist on the choline moiety which exhibit a pKa of 139 Therefore PC can engage strong interactions with the residual proteins fragments contained in the heavy phase The fatty acid composition of neutral oil and polar lipids obtained by solvent and proteolytic extractions compared to literature are shown in Table 5 As stated above the overall PUFA content was high and stood around 40 to 50 of total fatty acids in total lipids: 464 and 418 by solvent extraction and enzymatic treatment respectively compared to literature: 418 (Halilolu and others 2003) According to Shirai and others (2006) the polar lipid content was nearly 50 to be compared with the 534 It is noticeable that DHA reached 297 of total fatty acids in the polar lipids fraction This content was one of the highest DHA levels found in animals tissues unless in the heavy fraction resulting from salmon head proteolysis by Alcalase 24 L for example where the concentration of DHA is about 33 (Alder-Nissen 1977) Major phospholipids were represented by PC and PE in the solvent extract They appeared slightly different regarding their fatty acid composition For instance PC contained 23 palmitic acid compared with 114 in PE Despite that the overall PUFA content was the same (414 total fatty acids) EPA reached Extraction and Fractionation of PUFAs from Fish Using Supercritical CO2 Supercritical fluid extraction and fractionation of fish oil fatty acids have been studied by many researchers during the last 20 y (Esqvel and others 1997; Borch-Jensen and Mollerup 1999; Brunner and Riha 2000; Ltisse and others 2006; Perretti and others 2007) In general pressure temperature CO2 rate and time seemed to be important parameters for the optimization of extraction Several studies showed the influence of parameters which included pressure temperature CO2 rate and time Pressure temperature and time have been studied by zden (2000) pressure and temperature by Esqvel and others (1997) and Fleck and others (1998) while temperature pressure and CO2 rate have been studied by Dunford and others (1998) However fractionation of fish oil fatty acid ethyl esters was investigated with the aim of obtaining a lipid fraction enriched in -3 fatty acids and with a suitable EPADHA ratio Perretti and others (2007) reported the possibility of modifying the original fatty acid ethyl ester concentrations by optimizing the extraction conditions in terms of pressure temperature and supercritical carbon dioxide flow rate The authors conducted 2-h runs using different pressures (100 140 150 and 300 bar) and different liquid CO2 flow rates (25 35 5 and 10 kgh) keeping the temperatures of the 3 column sections at 40 50 and 60 C respectively starting from the bottom They stated that supercritical fluid fractionation appears to be a useful processing technique for changing the composition of lipids in order to obtain high-value functional products The use of proper fractionation temperatures and pressures along with the column influenced the solvent-to-feed ratio to obtain fractions with suitable composition for market requirements Dunford and others (1997) studied supercritical CO2 extraction of oil and residual proteins from Atlantic mackerel (Scomber scombrus) They reported that supercritical fluid extraction of fish muscle may be an alternative technique to produce high-quality fish meal and oil; and it has been used for fish processing The concentration of -3 fatty acids in fish oil using supercritical carbon dioxide (SC-CO2) was studied extensively by Eisenbach (1984) Krukonis (1988) Rizvi and others (1988) Nilsson and others (1988 1989) and Higashidate and others (1990) However oil extraction directly from high-fat fish muscle has attracted less attention than fish oil fractionation Extraction of ground freeze-dried Antarctic krill with SC-CO2 at 25 to 40 MPa and 40 to 80 C yielded oils which contained no phospholipids (PL) but triglycerides (TG) with an eicosapentaenoic acid (EPA) content of 11 (Yamaguchi and others 1986) Ikushima and others (1986) extracted freeze-dried mackerel (Scomber japonicus) powder using SC-CO2 at 49 to 245 MPa and 40 C The oil yield increased much with pressure compared to using 5-h hexane extraction In their study a kinetic model was developed to describe transport phenomena within the solids during the SCFE process SC-CO2 and SC-CO2ethanol (EtOH) mixtures have been used to remove lipids from trout muscle with a moisture content of 70 (ww) (Hardardottir and Kinsella 1988) Those authors reported that lipid removal did not improve when pressure and temperature were increased from 138 to 345 MPa and from 40 to 50 C probably due to the very high moisture content of the feed SC-CO2 and SC-CO2EtOH extraction removed 78 and 97 of the lipids and 97 and 99 of cholesterol respectively The moisture content of the SC-CO2-extracted muscle (131) was substantially lower than the initial moisture content of 749 indicating removal of moisture with the lipids (Hardardottir and Kinsella 1988) Temelli and others (1994 1995) investigated SC-CO2 extraction of freeze-dried Atlantic mackerel (Scomber scombrus) studying in particular the changes in proteins due to high-pressure oil extraction Extraction conditions of 345 MPa and 35 C resulted in high oil yield and minimal changes in residual proteins Water binding potential and pH of residual proteins changes in sarcoplasmic proteins and -3 fatty acid composition of the SC-CO2-extracted oil were reported In most of the cited studies (Ikushima and others 1986; Temelli and others 1994 1995) fish samples were freeze-dried prior to SC-CO2 extraction of oil to improve extraction efficiency Hardardottir and Kinsella (1988) froze trout muscle pieces in liquid nitrogen and ground them to increase surface area Moisture of the feed materials may interfere with SC-CO2 extraction of desired components from the sample matrix This was shown to be very important during the SC-CO2 extraction of lipids from muscle (King and others 1989) Extraction of intact muscle presents difficulties due to its fibrous structure and high moisture content (Wehling and others 1992) King and others (1989) reported higher extraction rates for meat products which were comminuted and dehydrated prior to SC-CO2 extraction of lipids However optimization of sample moisture content for SC-CO2 lipid extraction from muscle tissues and its effect on proteins has not been reported Correlation of moisture content with water activity in such biological samples should also be studied Water activity would be a critical factor affecting all component interactions during SC-CO2 extraction Dunford and others (1997) experimented with mackerel samples with moisture contents of 38 102 26 and 64 using SC-CO2 extractions at 35 C and 345 MPa for 5 h (CO2 flow rate was 22 04 gmin) The researchers characterized the lipid composition of SC-CO2 extracts and the residual oil They also analyzed changes in sarcoplasmic proteins after SC-CO2 extraction at different moisture contents using capillary electrophoresis (CE) Previous studies with Atlantic mackerel had shown that the highest -3 fatty acid concentration in the extract with minimal damage to proteins was attained under those conditions (Temelli and others 1994 1995) The amounts of extract obtained at 64 and 26 moisture were similar The amount extracted increased when the moisture content of mackerel samples was decreased to 102 from the original level of 64 prior to SC-CO2 extraction Further dehydration of samples from 102 to 38 did not increase the amount extracted with SC-CO2 The amount of oil extracted increased from 03 to 25 g when the moisture content was decreased from 64 to 26 However the extract yield was similar (25 to 27 g) for samples with 102 and 38 moisture Only 10 of the oil in the feed was extracted at the original moisture content (64 ww) The low oil recovery was due to 2 reasons: (a) high moisture content acted as a barrier to diffusion of SC-CO2 into and diffusion of oil out of the matrix and (b) the pasty consistency of chopped muscle samples reduced SC-CO2-sample contact These results indicated that it was not necessary to dry mackerel samples to less than 26 moisture to achieve higher oil yields from SC-CO2 extraction This would result in saving time (freeze drying is a very slow process) and energy Furthermore shorter drying times would reduce the risk of quality deterioration of proteins Corra and others (2008) extracted fish oil using CO2 and reported that -3 fatty acid has a better possibility of fractionation under the operational conditions ranging from 30615 to 32915 K and from 78 to 294 MPa This result indicates that the CO2 is more selective in the zone near its critical point to fractionate the triacylglycerols of fish oil However the solubility of 052 gkg CO2 obtained for the fish oil at 30115 K and 78 MPa was much lower that the solubility under the other operational conditions (Table 6) The low value for solubility can limit the process because of the high consumption of solvent Table 6 shows the solubility of the oil in SC-CO2 as a function of temperature and pressure or as a function of temperature and density Since the light phase of the equilibrium mixture for the systems under study was only slightly concentrated in the solute the density was considered to be that of pure CO2 and they were calculated by an empirical equation (Huang and others 1985) which reproduces the experimental values of IUPAC (Angus and others 1976) The results show that the solubility of the fish oil increased with increase in pressure at constant temperature the effect of temperature being less meaningful With an increase from 31315 to 32315 K at 196 MPa the solubility decreased by about 18 and the same increase in temperature at 294 MPa resulted in a decrease in solubility of only about 7 An increase from 30315 to 31315 K at 294 MPa and from 31315 to 32315 K at 245 MPa showed no variation in the values for solubility The highest values for solubility found in this study were about 7 g oilkg CO2 at 294 MPa and the different temperatures and higher values can be expected at higher pressures For Atlantic mackerel oil the highest solubility observed was 1421 gkg CO2 at 345 MPa and 30815 K (Temelli and others 1995) The behavior of the solubility values obtained in this study was consistent considering that the solubility of a solute is directly dependent on the density of the solvent and that increments in pressure at constant temperature promote an increase in the density of the solvent In the pressure range studied the solubility did not show a crossover point for retrograde behavior Knowledge of the solubility of the sample under study is important to estimate the economic viability of the process due to the fact that the greater the value for solubility the lower the consumption of solvent In general the solubility of oils can be estimated by correlations as proposed by Del Valle and Aguilera (1998) Corra and others (2008) also fractionated fish oil with babassu fat using SC-CO2 In order to better visualize how the fatty acid composition of the triacylglycerols the molar mass and the degrees of unsaturation influenced the selectivity of the solvent a mixture containing equal parts of fish oil and babassu fat was prepared and the fractionation analyzed The possibility of babassu fat to influence the equilibrium and the fractionation of fish oil was also analyzed Babassu fat was chosen because it contains a different fatty acid composition in comparison with the fish oil being composed of approximately 80 of saturated fatty acids (C8:0 to C18:0) and 20 of unsaturated C18:1 and C18:2 (Gioielli and others 1998) Corra and others (2008) also reported that the solubility of the mixture of fish oil and babassu fat (1 : 1) was higher than the solubility of the fish oil due to the content of shortchain fatty acids (lower molar mass) from the babassu fat This behavior can be verified in the literature (Soares and others 2007) To verify if the babassu fat has some influence on the separation of the compounds of interest the solubility of the fatty acids EPA and DHA in the extracts of fish oil and in the mixture of fish oil and babassu fat (1 : 1) were analyzed under the operational conditions of 78 MPa and 30115 K The extracts obtained under these distinctive situations resulted in triacylglycerols containing 117 and 145 of EPA corresponding to values for solubility of 0061 and 0032 g (EPA)kg CO2 In an analogous way they contained 1124 and 132 of DHA with solubility values corresponding to 0058 and 0029 g (DHA)kg CO2 confirming that the solubility values for the triacylglycerols containing EPA and DHA reduced to half the original value in the mixture (fish oil and babassu fat) This behavior indicates that the babassu fat did not promote a change in the fractionation of the fish oil However the researchers concluded that -3 fatty acid composition of the fish oil used in this study was high almost 13 of the total fatty acids and it was highly probable that the fatty acids of interest would be present in almost all the triglyceride molecules as evidenced by the difficulty in fractionating them Thus it is possible that it could be used to enrich fish oil containing a lower -3 fatty acid composition Several studies also on the supercritical extraction of fish oil and mainly the fractionation of EPA and DHA as fatty acid ethyl esters from fish oils are shown in Table 7 such as the SC-CO2 extraction of oil from sardine (Ltisse and others 2006); the influence of moisture from the matrix (Atlantic mackerel) on the oil yield and on the changes in sarcoplasmic proteins of the matrix after SC-CO2 extraction (Dunford and others 1997); the SC-CO2 fractionation of EPA and DHA as fatty acid ethyl esters (Riha and Brunner 1999; Alkio and others 2000; Jaubert and others 2001; Espinosa and others 2002; Gironi and Maschietti 2006; Jachmanin and others 2007; Perretti and others 2007); and the phase equilibrium for SC-CO2 and fish oil including free fatty acids cholesterol wax esters and di- and triacylglycerols (Borch-Jensen and Mollerup 1997) Future Development of Fish Oil Extraction Method Using SC-CO2 By far it is obvious that the SFE method is very advantageous and environmentally friendly over other conventional either solvent or enzyme extraction methods for recovering PUFAs but the major problem is the SFE method consumes a lot of CO2 To overcome this problem Zaidul and others (2007) introduced the pressure swing technique for the separation of palm kernel oil (PKO) from undehulled ground palm kernel using supercritical CO2 The pressurization of CO2 into the sample with holding to penetrate into the sample matrix and then depressurization is denoted as pressure swing (PS) technique Researchers applied the PS technique and were able to get higher yield with least amount of CO2 Extractions were performed at 3532 K and at pressures from 10 to 25 MPa Results were compared with continuous extractions in which supercritical CO2 was flowed through the packed bed of solids for a given time period For the PS extractions some intact or bound oil could be extracted from the 3rd PS step at 15 MPa while for continuous extractions pressures of 20 MPa were required to obtain comparable yields In the PS extractions disruption of the oil glands in palm kernel granules probably lead to higher yields obtained at 20 and 25 MPa and this was confirmed with SEM micrographs However almost all of the oil was extracted using combined PS and continuous extraction at 25 MPa The researchers developed a simple correlation based on the kinetic mass transfer model which allows one to estimate the minimum amount of CO2 required for a given yield However the model that was developed by Zaidul and others (2007) could be applied to extract and fractionate the fish oil especially PUFA (omega-3 6 fatty acids) with minimum amount of CO2 usage for a wide range of conditions and would provide the means for studying other pressure temperature and flow rates including temperature and pressure programming PUFA Production at Industrial-Scale by the Supercritical Fluid (SFC) Method Alkio and others (2000) reported a systematic procedure for developing supercritical fluid (SFC) separation method for producing EPA and DHA ethyl ester concentrates The development was done in preparative laboratory-scale batches using the specific production rate of EPA and DHA ethyl esters as the target function which was maximized The specific production rate was the hourly production in grams of the desired compound per kilogram of stationary phase Their experimental material was tuna oil which is a low-value by-product of the fishmeal industry A series of preparative SFC runs were carried out at estimated optimal conditions to obtain the real production rate They applied pure CO2 (without co-solvents) to study the technical and economic feasibility of producing EPA and DHA ethyl ester concentrates from by-product fish oil The fatty acids in the tuna oil were converted to ethyl esters by transesterification with absolute ethyl alcohol In the transesterification 50 g tuna fish oil was first dried with anhydrous Na2SO4 Its water content after drying was 024 wt Dried oil was filtered and refluxed for 15 h with 350 g absolute ethyl alcohol Freshly made sodium alcoholate was used as catalyst The mixture was then extracted with n-hexane to obtain the fatty acid esters The composition of the resulting fish oil ethyl ester mixture was analyzed by GCMS and GCFID After transesterification the DHA content of the oil was higher the EPA content unchanged and the oleic and palmitic acid contents lower than in the original oil The authors suspect that the repeated extraction of the ethanol containing aqueous layer with hexane did not remove all the lighter fatty acid esters However the slight change of the fatty acid composition during transesterification does not influence the SFC process development Alkio and others (2000) reported the most critical impurities in obtaining pure DHA were other C22 esters The separation between EPA and DHA was incomplete but due to the marginal amount of EPA in the starting material DHA did not interfere with DHA At all load ratio levels the 1st DHA fractions also contained 18:0 and 18:1 esters This indicates that the low unsaturated C18 esters tend to tail This was not observed with C20 esters or with C18 esters of a higher degree of unsaturation The separation of EPA from DHA judged feasible by Reichmann and Brunner (1996) was complete at each load ratio The calculated SFC separation factor between EPA and DHA was good 140 In the preparative runs the measured production rate at 90 (wt) purity was 085 g DHA-ethyl ester(kg stationary phase h) At 80 wt purity the production rate was 19 g DHA ethyl ester(kg stationary phase h) The purest EPA fractions at 5 and 25 gkg load ratios contained respectively 33 and 538 wt EPA Reducing the load to 125 gkg did not increase EPA purity Similarly to the preparation of DHA also in the separation of EPA the main impurities were stearic (18:0) and oleic (18:1) acid esters At 50 wt purity the specific production rate of EPA was 023 g(kg h) However the production of DHA- and EPA-ethyl ester concentrates from transesterified tuna oil at 80 to 95 and 50 wt respective purities requires only one supercritical chromatographic step The flowsheet of an industrial SFC process with descriptions of main equipment has been described by Aaltonen and others (1998) An SFC process which produces 1000 kg DHA-ethyl ester and 400 kg EPA-ethyl ester concentrates per year requires that 26 tons of CO2 per hour is circulated in the process The octadecylsilane-grafted silica (ODS) stationary phase requirement is 160 kg which would preferably be packed in four parallel 600-mm-id columns Major equipment for such an SFC process costs about US 2 million In assuming that the stationary phase would have to be replaced once a year the total SFC operating costs are US 550kg DHA and EPA concentrate The purification cost is sensitive to the lifetime of the stationary phase The cost almost equals the US200 to 500 range reported in 1994 by KD Pharma (Lembke and Engelhardt 1994) and is considerably less than the US4000DHA concentrate (95) reported by Shisheido Corp (Anonymous 1996) where a proprietary silver-containing stationary phase was used Conclusions The potentiality to obtain health benefits of PUFAs especially for the family of omega-3 fatty acids (linolenic acid arachidonic acid EPA and DHA) have been steadily increasing Those fatty acids have been identified to have a role in ameliorating various human diseases Marine fishes especially Indian mackerel offer higher amounts of PUFAs (EPA and DHA) compared to other marine fishes The extraction and purification methods for those fatty acids are summarized in this review Emphasis is given on recent advances in technological developments particularly supercritical fluid extraction (SFE) from marine fish especially from Indian mackerel and their fatty acid compositions Use of SFE technology that offers suitable extraction and fractionation appears to be promising for the production of omega-3 fatty acid concentrates for the food and pharmaceutical industries A brief overview is 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Omega-3 fatty acids in Lake Superior fish J Food Sci 55 : 71 3 Weaver BJ Holub BJ 1988 Health effects and metabolism of dietary eicosapentaenoic acid Prog Food Nutr Sci 12 : 111 50 Wehling RL Froning GW Cuppett LS Niemann L 1992 Extraction of cholesterol and other lipids from dehydrated beef using supercritical carbon dioxide J Agric Food Chem 40 : 1204 7 Yamaguchi K Murakami M Nakano H Konosu S Kokura T Yamamoto H Kosaka M Hata K 1986 Supercritical carbon dioxide extraction of oils from Antarctic krill J Agric Food Chem 34 : 904 7 Zaidul ISM Nik Norulaini NA Mohd Omar AK Sato Y Smith Jr RL 2007 Separation of palm kernel oil from palm kernel with supercritical carbon dioxide using pressure swing technique J Food Eng 81 : 419 28 ABSTRACT:Polyunsaturated fatty acids (PUFAs) especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are currently in demand in the pure form and actively being studied to understand their potential roles in human health Arachidonic acid 20:4 (n-6) and DHA 22:6 (n-3) are important in normal neurodevelopment and visual function Infants fed formula often have low blood lipid 20:4 (n-6) and 22:6 (n-3) Consumption of fish oils may increase the 20:5 (n-3) (EPA) and 22:6 (n-3) (DHA) in human blood Some marine fish oils contain higher amounts of arachidonic acid EPA and DHA PUFA contents in different marine fishes and methods for their extraction and fractionation in terms of fatty acid constituents in the form of methyl esters are covered in this review Emphasis is given to the fractionations of EPA and DHA by means of supercritical fluid extractions (SFE) The advantages of SFE compared to conventional methods are discussed in this review PUFAs are usually extracted at about 10 to 30 MPa and at 40 to 80 C SFE is a promising and currently the best technique to extract PUFAs especially EPA and DHA from marine and freshwater fish